The present invention relates to a novel detecting reagent for double-stranded nucleic acid and to a double-stranded nucleic acid detecting method employing it.
Gene expression analysis experiments using microarrays have conventionally employed the following experimental procedure. Specifically, (1) a probe nucleic acid is immobilized on a substrate, (2) two different biospecimens, for example, cells at different cell cycle stages, or an mRNA mixture prepared from healthy human tissue and patient tissue or a cDNA mixture prepared by reverse transcription therefrom, are labeled with dyes having different fluorescent properties to prepare target nucleic acid, and (3) the target is competitively hybridized to the probe and then washed, and the relative fluorescent intensities of the two different dyes bonded to the remaining target are measured.
The methods currently used for immobilization of the probe nucleic acid are largely of two types. That is, (1) methods in which a lithographic technique is used to polymerize the probe nucleic acid monomers onto the substrate one residue at a time, and (2) methods in which the specimen-derived nucleic acid sample, or nucleic acid prepared therefrom by PCR or reverse transcription, is spotted.
Several methods are used for labeling of the target nucleic acid, as well. These include (1) methods in which nucleotide substitution reaction is utilized for insertion of labeled nucleotides into the specimen-derived mRNA mixture, and (2) methods in which a labeled substrate is used for the specimen-derived mRNA mixture and the labeled nucleic acid is amplified by reverse transcription and PCR.
However, labeling by such methods can affect the expressed gene abundance ratio in the sample, and this can constitute a problem in terms of the detecting precision or detection sensitivity for specimens with a low abundance ratio in the measuring sample.
It has therefore been strongly desired to develop a method of detecting and quantifying double-stranded nucleic acid formed by hybridization with a probe, without carrying out any labeling of the target nucleic acid.
It is an object of the present invention to provide a novel detecting reagent for double-stranded nucleic acid which requires absolutely no labeling of the target nucleic acid, as well as a double-stranded nucleic acid detecting method employing it.
As a result of diligent research directed toward solving the aforementioned problems associated with the prior art, the present inventors have completed the present invention upon successfully discovering a detecting reagent which (1) intercalates only with double-stranded nucleic acid formed by target nucleic acid, without any labeling, and (2) has a function allowing high-precision, high-sensitivity fluorescent analysis.
Specifically, the detecting reagent for double-stranded nucleic acid according to the invention is characterized by comprising, in the same molecule, a naphthalenediimide skeleton as a group which is intercalatable into double-stranded nucleic acid, and a xcex2-diketone group having a specific structure rendering it capable of forming a lanthanoid metal complex.
More specifically, the detecting reagent for double-stranded nucleic acid according to the invention is characterized by having a chemical structure represented by the following formula (1) or (2). 
where R is a linker represented by xe2x80x94CmH2mN(Rxe2x80x2)CnH2nxe2x80x94, Rxe2x80x2 is an alkyl group, and m and n are each integers of 1-10.
The detecting reagent for double-stranded nucleic acid according to the invention also encompasses those comprising a fluorescent lanthanoid metal complex wherein a lanthanoid metal is coordinated in the xcex2-diketone group.
The double-stranded nucleic acid detecting method of the invention is characterized by forming double-stranded nucleic acid with target nucleic acid and a probe, intercalating into the double-stranded nucleic acid a detecting reagent for double-stranded nucleic acid comprising a lanthanoid metal complex according to the invention, and measuring the fluorescence of the lanthanoid metal complex to detect and quantify the double-stranded nucleic acid. The invention also includes a method of quantifying the molecular length and number of molecules of the target nucleic acid by this detecting method, and a method of determining the base sequence of the nucleic acid. The invention still further includes this detecting reagent for double-stranded nucleic acid which is bonded to molecules in a specimen such as various other organic compounds, nucleic acids, oxygen, antigens, antibodies, etc., as well as a method of detecting nucleic acid and proteins using it.
The present invention will be explained hereunder in further detail by way of examples, with the understanding that they are only intended to be illustrative and not restrictive on the invention.